# US 9,005,929 B2 · Multibase delivery for long reads in sequencing by synthesis protocols

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- Number: US 9,005,929 B2
- Document ID: US9005929B2
- Status: Granted
- Kind: B2
- Application number: 14/200,674
- Filed: 2014-03-07
- Published: 2014-09-11
- Granted: 2015-04-14
- Earliest claimed: 2008-12-23
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Mostafa Ronaghi; Helmy A. Eltoukhy
- Web page: https://patentgrep.com/patent/US9005929B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for obtaining nucleic acid sequence information that can include steps of (a) providing a first sequencing reagent to a target nucleic acid, wherein the first sequencing reagent comprises at least two different nucleotide monomers, (b) detecting the incorporation of a nucleotide monomer present in the first sequencing reagent into a polynucleotide strand complementary to at least a portion of the target nucleic acid, (c) providing a second sequencing reagent to said target nucleic acid, wherein the second sequencing reagent comprises one or more nucleotide monomers, at least one of the one or more nucleotide monomers being different from the nucleotide monomers present in the first sequencing reagent, and wherein the second sequencing reagent is provided subsequent to providing the first sequencing reagent, and (d) detecting the incorporation of a nucleotide monomer present in the second sequencing reagent into the polynucleotide strand.

## Claims

### Claim 1

A method for obtaining nucleic acid sequence information, said method comprising the steps of:
(a) providing a first sequencing reagent to a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;
(b) detecting the incorporation of a nucleotide monomer present in said first sequencing reagent into a polynucleotide strand complementary to at least a portion of said target nucleic acid;
(c) providing a second sequencing reagent to said target nucleic acid, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent, and wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent;
(d) detecting the incorporation of a nucleotide monomer present in said second sequencing reagent into the polynucleotide strand; and
(e) repeating steps (a) through (d) for said target nucleic acid, thereby obtaining sequence information for at least a portion of said target nucleic acid.

### Claim 2

A method for obtaining nucleic acid sequence information, said method comprising the steps of:
(a) detecting the incorporation of a nucleotide monomer present in a first sequencing reagent into a polynucleotide strand complementary to at least a portion of a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;
(b) removing at least a portion of said first sequencing reagent; and
(c) detecting the incorporation of a nucleotide monomer present in a second sequencing reagent into the polynucleotide strand, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent; and
(d) repeating steps (a) through (c) for said target nucleic acid, whereby sequence information for at least a portion of said target nucleic acid is obtained.

### Claim 3

A method for obtaining nucleic acid sequence information, said method comprising the steps of:
(a) detecting the incorporation of a nucleotide monomer present in a first sequencing reagent into a first polynucleotide strand complementary to at least a portion of a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;
(b) detecting the incorporation of a nucleotide monomer present in a second sequencing reagent into the first polynucleotide strand, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent;
(c) removing said first polynucleotide strand; and
(d) detecting the incorporation of a nucleotide monomer present in a third sequencing reagent into a second polynucleotide strand complementary to at least a portion of said target nucleic acid, wherein said third sequencing reagent comprises two or more nucleotide monomers, wherein at least one of said two or more nucleotide monomers is different from the at least two different nucleotide monomers present in said first sequencing reagent and at least one of said two or more nucleotide monomers is different from the one or more different nucleotide monomers present in said second sequencing reagent; and
(e) repeating steps (a) through (d) for said target nucleic acid.

### Claim 4 (depends on 1)

The method of claim 1, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

### Claim 5 (depends on 4)

The method of claim 4, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

### Claim 6 (depends on 1)

The method of claim 1, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

### Claim 7 (depends on 6)

The method of claim 6, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

### Claim 8 (depends on 1)

The method of claim 1, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

### Claim 9 (depends on 8)

The method of claim 8, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

### Claim 10 (depends on 1)

The method of claim 1, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

### Claim 11 (depends on 10)

The method of claim 10, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

### Claim 12 (depends on 11)

The method of claim 11, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.

### Claim 13 (depends on 2)

The method of claim 2, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

### Claim 14 (depends on 13)

The method of claim 13, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

### Claim 15 (depends on 2)

The method of claim 2, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

### Claim 16 (depends on 15)

The method of claim 15, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

### Claim 17 (depends on 2)

The method of claim 2, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

### Claim 18 (depends on 17)

The method of claim 17, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

### Claim 19 (depends on 2)

The method of claim 2, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

### Claim 20 (depends on 19)

The method of claim 19, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

### Claim 21 (depends on 20)

The method of claim 20, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.

### Claim 22 (depends on 3)

The method of claim 3, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

### Claim 23 (depends on 22)

The method of claim 22, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

### Claim 24 (depends on 23)

The method of claim 23, wherein said third sequencing reagent comprises two nucleotide monomers, and wherein one of said two nucleotide monomers is different from the at least two nucleotide monomers present in the first sequencing reagent.

### Claim 25 (depends on 3)

The method of claim 3, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

### Claim 26 (depends on 25)

The method of claim 25, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

### Claim 27 (depends on 26)

The method of claim 26, wherein said third sequencing reagent comprises three nucleotide monomers, and wherein one of said three nucleotide monomers is different from the at least two nucleotide monomers.

### Claim 28 (depends on 3)

The method of claim 3, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

### Claim 29 (depends on 28)

The method of claim 28, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

### Claim 30 (depends on 3)

The method of claim 3, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

### Claim 31 (depends on 30)

The method of claim 30, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

### Claim 32 (depends on 31)

The method of claim 31, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.